The present invention is related to a newly discovered and isolated human chorionic peptidase (C-ase-1) and its uses, such as affecting states of pregnancy and as a drug screening tool.
Gonadotropin releasing hormone-like material has been identified in various biological fluids and tissues such as hypothalami, semen, testes, placenta, pancreas and mammary carcinoma. It is generally believed that gonadotropin releasing hormone (GnRH) is identical or similar to luteinizing hormone releasing factor (LRF) and is a small peptide. This small peptide is generally agreed to be a decapeptide with the amino acid sequence: pyro-glu-his-trp-ser-tyr-gly-leu-arg-pro-gly-NH.sub.2. GnRH-like material appears to be synthesized by placenta and numerous other tissues and to have receptor sites in diverse organs. The present invention describes a novel protein, a human chorionic peptidase enzyme (C-ase-1) which has never before been identified and is completely unique in many ways from GnRH. Antibodies directed toward GnRH have indicated the presence of GnRH-like material at numerous other biological sites. A 92 amino acid peptide has been reported which contains the decapeptide GnRH (Seeburg et al., (1984) Nature, 311:666), but the human chorionic peptidase (C-ase-1) described herein differs from it.
Earlier studies by Siler-Khodr et al. ((1982) Science, 207:315) and others have demonstrated that an immunologically and biologically active GnRH was synthesized and released from human placental extracts. This GnRH eluted from CM-cellulose in the same area as synthetic GnRH. In addition, Lee at al. (1981) (Acta Endocrinol., 96:394) reported an apparent GnRH immunoreactivity from acid extracts of placentas that eluted on HPLC in the area of GnRH. These findings led to the hypothesis that placental apparent GnRH immunoreactivity and synthetic GnRH were chemically similar. Additionally, Tan et al. (1982) (Biochem Biophys. Res. Commun., 109:106 1) reported that the GnRH decapeptide sequence was present in the acid extracts of placenta. More recently, Seeburg et al. (1984) (Nature, 311:666), using cDNA expression, deciphered an mRNA coding for 92 amino acids in which the GnRH sequence was contained.
Certainly, the presence of the decapeptide GnRH in the placenta has been firmly supported. The role of GnRH in placental endocrinology has not been answered by the above cited studies. It has been demonstrated that synthetic GnRH can affect placental hormonogenesis, yet high concentrations are needed. An antagonist of GnRH has been shown to inhibit placental hormonogenesis both in vitro (Siler-Khodr et al., (1983) Life Sci., 32:2742) and in vivo (Siler-Khord et al., (1984) Fertil-Steril, 41:448). Other studies have demonstrated that a placental receptor recognizes synthetic GnRH; however, the dissociation constant of the placental receptor for synthetic GnRH was found to be only 10.sup.-7 M, and a potent agonist of GnRH on the pituitary had no greater affinity for this placental receptor than did GnRH. Thus, it appeared that the placental receptor recognizing synthetic GnRH differed from the pituitary receptor. The inventor has also noted that, although there was newly synthesized apparent GnRH immunoreactivity recovered in the GnRH area following CM cellulose chromatography, it accounted for only &lt;1% of the apparent GnRH immunoreactivity. In addition, as reported herein, it was observed that apparent GnRH immunoreactivity of the placenta was extracted with a very low yield with methods typically utilized for extraction of hypothalamic GnRH, i.e. acid or methanol.
Applicant was able to extract from human placenta with an aqueous neutral buffer a substantially purified preparation of a chorionic protein with apparant GnRH-immunoreactivity and prostaglandin and hCG-releasing activity, and characterized it as a large molecular weight glycoprotein, having a molecular weight of between about 50,000 and about 70,000 daltons. It was found that this factor possessed the ability to inactivate GnRH. However, this preparation was purified only about 10% from crude placental extracts. Additionally, the mechanism by which the factors apparent immunoreactivity to GnRH was not known. It was hypothesized that the factors apparent immunoreactivity to GnRH was either a function of the factors immunological competition with or direct alteration of GnRH. This alteration was thought to be due to a hydrolytic degradation or some other unspecified activity. These findings led the inventors to consider whether this factor could be used to specifically regulate the synthesis, release, and metabolism of the myriad of hypothylamic-like releasing/inhibiting hormones produced by or circulating in the human placenta (i.e., GnRH, angiotensin II, TRH, somatostatin). Further, it was also speculated that the specificity of the factors action could be more closely regulated in vivo if the factor were further purified and specific enzyme inhibitors thereto characterized.
As with any partially purified preparation, the possibility that the crude preparation would elicit an antigenic response in the treated animal existed. Additionally, such a risk was amplified by the relatively low level of purification obtained in prior preparations, thus requiring large amounts of the impure preparation be administered in order to achieve desired doses of the "factor". Moreover, the elucidation of the mechanisms by which the active ingredient precipitated the degradation of GnRH and other factors would allow for the selection and use of the most effective enzyme analogues to control chorionic GnRH, as well as other factors' activity at the placental level. Such information would also serve in the regulation of other chorionic tissues where GnRH acts in a paracrine fashion to regulate hormonal functions during pregnancy. Thus, the further characterization and purification of a placental enzyme isolate which was non-antigenic to the host, possessed high specific activity and which specifically inhibited peptides used clinically during pregnancy would be a significant step forward in the medical science of animal reproduction and growth.
The following comprises a list of abbreviated terms as used throughout the present application.
C-ase-1--chorionic peptidase one. PA1 OXY--oxytocin. PA1 TRH--thyrotropin-releasing factor. PA1 GnRH--gonadotropin-releasing hormone. PA1 irGnRH--immunoreactive gonadotropin-releasing hormone. PA1 hCGRF--human chorionic gonadotropin-releasing factor. PA1 DTT--dithiothreitol. PA1 CRF--corticotropin-releasing factor. PA1 Try-CRF--tryptophan corticotropin-releasing factor. PA1 LH--luteinizing hormone. PA1 rLH--rat luteinizing hormone. PA1 FSH--follicle stimulating hormone. PA1 rFSH--rat follicle stimulating hormone. PA1 LRF--luteinizing hormone-releasing factor. PA1 EDTA--ethylenediaminetetraacetic acid. PA1 hCG--human chorionic gonadotropin. PA1 hCS--human chorionic somatomammotrophin. PA1 BSA--bovine serum albumin. PA1 PGE--prostaglandin E. PA1 MPF--metabolite prostaglandin F.sub.2 alpha; PA1 SRIF--somatotropin release-inhibiting factor; somatostatin. PA1 Try-SRIF--tryptophan somatotropin release-inhibiting factor. PA1 MSH--melanocyte-stimulating hormone.